Test piece and system for reading out image information from the test piece

ABSTRACT

A test piece for use in biological analyses includes a plurality of different known specific binding substances disposed in predetermined positions on a substrate. The specific binding substances are disposed on a plurality of surfaces provided by the substrate and arranged in the direction of thickness of the substrate.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to a test piece for use in DNA analysis,immunological analysis, and the like, and a system for reading out imageinformation from the test piece.

Description of the Related Art

Recently, genetic engineering has exhibited rapid progress, and thehuman genome project for decoding the base sequence of human genomeswhich amount to 100,000 in number is progressing.

Further, enzyme immunoassay, fluorescent antibody technique and the likeutilizing antigen-antibody reactions have been used in diagnoses andstudies, and studies for searching DNAs which affect genetic diseasesare now progressing. In such a situation, a microarray technique is nowattracting attention.

In the microarray technique, a microarray chip (sometimes called a DNAchip) comprising a plurality of known cDNAs (an example of specificbinding substances) coated in a matrix on a substrate such as a membranefilter or a slide glass at a high density (at intervals of not largerthan several hundred μm) is used and DNAs (an example oforganism-originating substances) taken from cells of a normal person Aand labeled with a fluorescent dye a and DNAs taken from cells of agenetic-diseased person B and labeled with a fluorescent dye b aredropped onto the microarray chip by pipettes or the like, therebyhybridizing the DNAs of the specimens with the cDNAs on the microarraychip. Thereafter, exciting light beams which respectively excite thefluorescent dyes a and b are projected onto the cDNAs by causing theexciting light beams to scan the microarray chip and fluorescenceemitted from each other of the cDNAs is detected by a photodetector.Then the cDNAs with which the DNAs of each specimen are hybridized aredetermined on the basis of the result of the detection, and the cDNAswith which the DNAs of the normal person A are hybridized and those withwhich the DNAs of the diseased person B are hybridized are compared,whereby DNAs expressed or lost by genetic disease can be determined.

In the microarray technique, it is necessary to preciselytwo-dimensionally scan the microarray chip coated with cDNAs at a highdensity, and there has been proposed a radiation image read-outapparatus with such a precise scanning system. See, for instance,Japanese Unexamined Patent Publication No. 10(1998)-3134.

The kinds of cDNAs to be used sometimes amount to several tens ofthousands and in such a case, the cDNAs must be coated on a plurality ofsubstrates. However when the number of the microarray chips to be usedincreases, replacement of microarray chips becomes troublesome.

SUMMARY OF THE INVENTION

In view of the foregoing observations and description, the primaryobject of the present invention is to provide a test piece on which anincreased number of specific binding substances such as cDNAs can bedisposed, and a system for reading out image information from the testpiece.

In accordance with one aspect of the present invention, there isprovided a test piece such as a microarray chip for use in biologicalanalyses comprising a plurality of different known specific bindingsubstances such as cDNAs disposed in predetermined positions on asubstrate such as a slide glass, wherein the improvement comprises that

the specific binding substances are disposed on a plurality of surfacesprovided by the substrate and arranged in the direction of thickness ofthe substrate.

The plurality of surfaces provided by the substrate and arranged in thedirection of thickness of the substrate may be opposite sides of thesubstrate or may be provided by a multi-layered substrate formed by aplurality of substrates which are stacked and bonded together so thatthe surfaces on which the specific binding substances are disposed aresubstantially in parallel to each other.

It is preferred that the specific binding substances be disposed on thesurfaces in positions where the specific binding substances on therespective surfaces do not interfere with each other in the direction ofthickness of the substrate, that is, the specific binding substances onthe respective surfaces do not overlap with each other in the directionof thickness of the substrate.

The substrate may be formed of any material so long as the specificbinding substances can be spotted and stably held on the substrate andthe substrate is optically transparent to the exciting light and thefluorescence emitted from the specific binding substances upon exposureto the exciting light. For example, the substrate may be a membranefilter or a slide glass. Further the substrate may be subjected topretreatment so that the specific binding substances are stably held onthe substrate.

The specific binding substances include hormones, tumor markers,enzymes, antibodies, antigens, abzymes, other proteins, nucleic acids,cDNAs, DNAs, RNAs, and the like, and means those which can bespecifically bound with an organism-originating substance. The means ofthe expression “known” differs by the specific binding substance. Forexample, when the specific binding substance is a nucleic acid, “known”means that the base sequence, the lengths of the bases and the like areknown, and when specific binding substance is protein, “known ” meansthat the composition of the amino acid is known. The specific bindingsubstances are disposed by one kind for each position.

In accordance with another aspect of the present invention, there isprovided a system for reading out image information from the test pieceof the present invention comprising

a test piece holder portion which holds a test piece of the presentinvention the specific binding substances on which have been hybridizedwith an organism-originating substance labeled with fluorescent dye,

an exciting light source which emits exciting light for exciting thefluorescent dye,

a photoelectric read-out means which photoelectrically reads outfluorescence emitted from the fluorescent dye upon exposure to theexciting light,

a scanning means which has an optical head for projecting the excitinglight onto the test piece and leading fluorescence, which is emittedfrom the fluorescent dye and travels through the surface of the testpiece onto which the exciting light is projected, to the photoelectricread-out means, and causes the exciting light to scan the test piece,and

a controller which controls the exciting light source, the photoelectricread-out means and the scanning means so that fluorescence emitted fromthe specific binding substances upon exposure to the exciting light isdetected for each of the surfaces of the test piece.

The test piece holder portion may comprise a table on which the testpiece is placed. In this case, the test piece is placed on the tablewith its one side in contact with the table, and accordingly, it isnecessary that the table is transparent to at least the fluorescence.When the test piece holder portion is in the form of a member whichsupports only the four corners of the test piece, the test piece holderportion need not be transparent.

The organism-originating substance may be a wide variety of substancesoriginated from an organism including hormones, tumor markers, enzymes,proteins, antibodies, various substances which can be antigens, nucleicacids, cDNAs, mRNAs and the like.

The exciting light is light suitable for exciting the fluorescent dyeincluding a laser beam.

As the photoelectric read-out means, a photomultiplier which can detectat a high sensitivity weak light such as fluorescence may be suitablyused. However, various known photoelectric read-out means such as acooled CCD may be used without limited to the photomultiplier.

In accordance with the present invention, since the specific bindingsubstances are disposed on a plurality of surfaces provided by thesubstrate and arranged in the direction of thickness of the substrate,an increased number of specific binding substances can be disposed onone test piece and accordingly, the number of test pieces to be used canbe less even if a large number of specific binding substances are used,whereby the frequency at which the test pieces are replaced can bereduced and reading operation can be effectively performed.

When the specific binding substances are disposed on opposite sides of asingle substrate, the test piece can be manufactured at low cost.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a test piece in accordance with a firstembodiment of the present invention,

FIG. 2 is a cross-sectional view taken along line I—I in FIG. 1,

FIG. 3 is a cross-sectional view of a test piece in accordance with asecond embodiment of the present invention,

FIG. 4A is a perspective view of an image information read-out system inaccordance with a third embodiment of the present invention,

FIG. 4B is a schematic side view of the image information read-outsystem,

FIG. 5 is a view for illustrating the operation of the image informationread-out system of the third embodiment,

FIG. 6A is a perspective view of an image information read-out system inaccordance with a fourth embodiment of the present invention, and

FIG. 6B is a schematic side view of the image information read-outsystem.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In FIGS. 1 and 2, a test piece 1 in accordance with a first embodimentof the present invention comprises a substrate 2 which is a slide glassin this particular embodiment, and a plurality of different cDNAsdisposed in a plurality of positions on opposite sides (upper and lowersides) of the substrate 2. The base sequences of the cDNAs are known andcorrespond to different DNAs. The kind of each cDNA and the position ofeach cDNA are predetermined.

As shown in FIG. 2, the cDNAs on the upper side of the substrate 2 andthose on the lower side of the substrate 2 are positioned not to overlapeach other in the direction of thickness of the substrate 2. Further theupper and lower sides of the substrate 2 have been subjected to surfacetreatment so that the cDNAs are bonded to the surfaces and accordingly,the cDNAs on the lower side of the substrate 2 cannot peel off thesubstrate 2. The thickness of the substrate 2 is about 1 mm, and each ofthe cDNAs is disposed on the surface of the substrate 2 in a spot of adiameter 30 to 100 μm with the spot intervals of about 300 μm.

FIG. 3 shows a test piece in accordance with a second embodiment of thepresent invention. The test piece shown in FIG. 3 is provided with asubstrate formed of a pair of substrate segments 2A and 2B which arebonded together with a spacer 3 interposed therebetween. The cDNAs aredisposed on the upper surfaces of the respective substrate segments 2Aand 2B not to overlap each other in the direction of thickness of thesubstrate segments 2A and 2B. The spacer 3 may either be discontinuousor continuous over the entire periphery of the substrate segments 2A and2B.

FIGS. 4A and 4B show an image information read-out system in accordancewith a third embodiment of the present convention for reading out imageinformation from the test piece 1 shown in FIG. 1. The image informationread-out system comprises a transparent sample table 20 on which thetest piece 1, having applied with an organism-originating substancelabeled with a fluorescent dye, is supported at its four corners, alaser 30 which emits a laser beam L in a wavelength band exciting thefluorescent dye, a lens 31 which converges the laser beam L as emittedfrom the laser 30 into a thin laser beam, a photomultiplier 40 whichphotoelectrically detects fluorescence K1 and K2 emitted from the cDNAsupon exposure to the laser beam L (K1 represents the fluorescenceemitted from the cDNAs on the upper surface of the substrate 2 and K2represents the fluorescence emitted from the cDNAs on the lower surfaceof the substrate 2), an optical head 50 which causes the laser beam L toimpinge upon the test piece 1 on the sample table 20 and leads thefluorescence K1 or K2 to the photomultiplier 40, a laser beam cut filter41 disposed on the optical path between the optical head 50 and thephotomultiplier 40, a condenser lens 55 which is disposed between thetest piece 1 and the photomultiplier 40 and forms a confocal opticalsystem together with a lens 53, an aperture plate 56 which has anaperture 56 a which permits to impinge upon the lens 41 only light froma portion of the test piece 1 on which the laser beam L is converged bythe lens 53, a main scanning system 60 which moves the optical head 50in the direction of arrow X at a constant speed, a sub-scanning means 80which moves the laser 30, the optical head 50, the condenser lens 55,the aperture plate 56, the laser beam cut filter 41 and thephotomultiplier 40 in the direction of arrow Y (perpendicular to thedirection of arrow X) integrally with each other, a logarithmicamplifier 42 which logarithmically amplifies a detecting signal outputfrom the photomultiplier 40, and an A/D converter 43 which digitizes theamplified detecting signal.

The laser 30 is arranged to emits the laser beam L in the direction ofarrow X, and the photomultiplier 40 is arranged to detect thefluorescence K1 or K2 impinging thereupon in the direction of arrow X.

The optical head 50 comprises a plane mirror 51 which reflects the thinlaser beam L, traveling in the direction of arrow X, in a directionperpendicular to the surfaces of the test piece 1, a mirror 52 which isprovided with an aperture 52 a through which the laser beam L reflectedby the plane mirror 51 impinges upon the test piece 1 and reflects themajor parts of the fluorescence K1 or K2, emitted downward from thelower surface of the test piece 1, to impinge upon the photomultiplier40, and the lens 53 which collimates the fluorescence K1 or K2 whichemits downward from the test piece 1 as divergent light. The planemirror 51, the mirror 52 with the aperture 52 a and the lens 53 areintegrated into a unit. The lens 53 is movable in the direction ofthickness of the test piece 1 or in the direction of arrow Z to move afocal point of the lens 53 selectively to the upper surface of thesubstrate 2 and to the lower surface of the same. When the fluorescenceK1 from the upper surface of the test piece 1 is to be detected, thefocal point of the lens 53 is moved to the upper surface of the testpiece 1 and when the fluorescence K2 from the lower surface of the testpiece 1 is to be detected, the focal point of the lens 53 is moved tothe lower surface of the test piece 1, whereby the florescence K1 and K2can be collimated to beams of substantially the same diameters.

The laser beam cut filter 41 is a filter which transmits thefluorescence K1 and K2 but does not transmit the laser beam L so that apart of the laser beam L scattered by the test piece 1, the sample table20 and the like cannot impinge upon the photomultiplier 40.

Operation of the image information read-out system of this embodimentwill be described, hereinbelow.

The position of the lens 53 is first adjusted so that the focal point ofthe lens 53 is on the upper surface of the substrate 2. Then the mainscanning means 60 moves the optical head 50 at a constant high speed inthe direction of arrow X. At each moment during movement of the opticalhead 50, the laser 30 emits a laser beam L in the direction of arrow Xand the lens 31 converges the laser beam L into a thin laser beam. Thethin laser beam L enters the optical head 50. The laser beam L is thenreflected upward by the plane mirror 51 and impinges upon a fine area onthe upper surface of the test piece 1 through the aperture 52 a of themirror 52 and the lens 53.

When an organism-originating substance labeled with fluorescent dyeexists in the fine area exposed to the laser beam L, the fluorescent dyeis excited by the laser beam L and emits fluorescence K1.

The fluorescence K1 spread around the area and the part of thefluorescence K1 traveling downward from the lower surface of the testpiece 1 is collimated by the lens 53 of the optical head 50 into asubstantially parallel downward beam and impinges upon the mirror 52.Though the part of the fluorescence K1 impinges upon the aperture 52 atravels further downward through the aperture 52 a (the diameter of theaperture 52 a is sufficiently small as compared with the beam diameter),the major part of the fluorescence K1 is reflected by the mirror 52 totravel in the direction of arrow X and to impinge upon thephotomultiplier 40 through the condenser lens 55, the aperture 56 a ofthe aperture plate 56 and the laser bean cut filter 41.

Though a part of the laser beam L impinging upon the test piece 1 isscattered by the test piece 1, the sample table 20 and the like andtravels toward the photomultiplier 40, it is prevented from impingingupon the photomultiplier 40 by the laser beam cut filter 41. Furthersince the test piece 1 and the photomultiplier 40 are opticallyconnected by a confocal optical system, fluorescence from a part of thetest piece other than the part exposed to the laser beam L is preventedfrom impinging upon the photomultiplier 40 and blur of a fluoresce imageobtained can be avoided even if the area exposed to the laser beam L isshifted or enlarged.

The fluorescence K1 impinging upon the photomultiplier 40 isphotoelectrically detected by the photomultiplier 40 and read out as anelectric signal. The electric signal is amplified by the amplifier 42and is converted to a digital signal by the A/D converter 43.

During these steps, the optical head 50 is kept moved in the directionof arrow X by the main scanning system 60, and a digital signal isoutput from the A/D converter 43 for each main scanning position on thetest piece 1.

Each time the main scanning along one line is ended, the sub-scanningmeans 80 slightly moves the laser 30, the optical head 50, the laserbeam cut filter 41 and the photomultiplier 40 in the direction of arrowY (sub-scanning) and the main scanning is repeated. The sub-scanning maybe effected in parallel to the main scanning.

Thus the entire area of the upper surface of the test piece 1 istwo-dimensionally scanned by the laser beam L, and image informationrepresenting the distribution of the organism-originating substanceslabeled by the fluorescent dye on the upper surface of the substrate 2is obtained.

Thereafter the optical head 50 is returned to the initial position bythe main scanning means 60 and the sub-scanning means 80. Then the lens53 is moved downward by d0 (FIG. 5) so that the focal point of the lens53 is on the lower surface of the substrate 2. Then the fluorescence K2emitted from the lower surface of the test piece 1 is detected andconverted to a digital signal in the same manner as described above andimage information representing the distribution of theorganism-originating substances labeled by the fluorescent dye on thelower surface of the substrate 2 is obtained.

The image information representing the distribution of theorganism-originating substances labeled by the fluorescent dye on theupper and lower surfaces of the substrate 2 is displayed on a monitor(not shown).

Thus in the image information read-out system of this embodiment, imageinformation can be read out from opposite sides of the test piece 1 ofthe first embodiment of the present invention, where cDNAs are disposedon opposite sides of the substrate 2.

The image information read-out system of this embodiment can be used toread out image information from the test piece of the second embodimentof the present invention shown in FIG. 3. In this case, the lens 53 ismoved so that the focal point of the lens 53 is selectively moved to theupper surface of the substrate segment 2A and that of the substratesegment 2B.

Though, in the embodiment described above, the fluorescence K1 emittedfrom the cDNAs on the upper surface of the substrate 2 and thefluorescence K2 emitted from the cDNAs on the lower surface of thesubstrate 2 are separately detected by moving the focal point of thelens 53 forming a confocal optical system, it is possible to separatelydetect the fluorescence K1 and the fluorescence K2 by moving theaperture plate 56 along the optical axis with the lens 53 keptstationary. That is, when the laser beam L is projected onto the testpiece 1 with the focal point of the lens 53 set at the middle betweenthe upper and lower surfaces of the substrate 2, the fluorescence K1 isemitted from the cDNAs on the upper surface of the substrate 2 and thefluorescence K2 is emitted from the cDNAs on the lower surface of thesubstrate 2. Depending on the position of the aperture plate 56 alongthe optical axis, only one of the fluorescence K1 and the fluorescenceK2 can pass through the aperture 56 a in the aperture plate 56.

FIGS. 6A and 6B show an image information read-out system in accordancewith a fourth embodiment of the present invention for reading out imageinformation from the test piece 1 shown in FIG. 1. The image informationread-out system of this embodiment comprises a sample table 20, a firstlaser 30A, a first lens 31A, a first photomultiplier 40A, an opticalhead 50, a first laser beam cut filter 41A, a first condenser lens 55A,a first aperture plate 56A, a main scanning system 60, a sub-scanningmeans 80, a first logarithmic amplifier 42A, and a first A/D converter43A, which are basically the same as the sample table 20, the laser 30,the lens 31, the photomultiplier 40, the optical head 50, the laser beamcut filter 41, the condenser lens 55, the aperture plate 56, the mainscanning system 60, the sub-scanning means 80, the logarithmic amplifier42, and the A/D converter 43 employed in the third embodiment. The imageinformation read-out system of this embodiment further comprises asecond laser 30B, a second lens 31B, a second condenser lens 55B, asecond aperture plate 56B, a second laser beam cut filter 41B, a secondphotomultiplier 40B, a second logarithmic amplifier 42B, a second A/Dconverter 43B, a polarization beam splitter 62 which transmits the laserbeam L1 emitted from the first laser 30A and reflects the laser beam L2emitted from the second laser 30B, and a half-silvered mirror 63 whichtransmits a part of the fluorescence K1 and the fluorescence K2 toimpinge upon the first photomultiplier 40A, and reflects the other partof the fluorescence K1 and the fluorescence K2 to impinge upon thesecond photomultiplier 40B.

The first and second lenses 31A and 31B are identical to each other. Thefirst and second lasers 30A and 30B are basically identical to eachother except that the laser beam LI emitted from the first laser 30A ispolarized in the vertical direction as seen in FIG. 6B and the laserbeam L2 emitted from the second laser 30B is polarized in a directionperpendicular to the surface of the paper on which FIG. 6B is drawn.With this arrangement, the laser beam L1 transmits the polarization beamsplitter 62 and the laser beam L2 is reflected by the same.

The distance d2 between the beam radiating end of the 16 second laser30B and the second lens 31B is set larger than the distance d1 betweenthe beam radiating end of the first laser 30A and the first lens 31A sothat the diameter of the laser beam L1 on the upper surface of thesubstrate 2 becomes equal to the diameter of the laser beam L2 on thelower surface of the substrate 2.

Further, the distance D2 between the second condenser lens 55B and thesecond aperture plate 56B is set larger than the distance D1 between thefirst condenser lens 55A and the first aperture plate 56A. In the fourthembodiment, the leaser beams L1 and L2 are simultaneously emitted fromthe first and second lasers 30A and 30B, and the fluorescence K1 and thefluorescence K2 emitted respectively from the upper and lower surfacesof the test piece 1 are simultaneously detected. The fluorescence K1 andthe fluorescence K2 emitted respectively from the upper and lowersurfaces of the test piece 1 simultaneously travel in the direction ofarrow X. The fluorescence K1 and the fluorescence K2 emittedrespectively from the upper and lower surfaces of the test piece 1 areseparated by the half-silvered mirror 63 to parts which respectivelytravel to the first and second photomultipliers 40A and 401B. Either ofthe parts includes both the fluorescence K1 and the fluorescence K2, andaccordingly, the distance D2 between the second condenser lens 55B andthe second aperture plate 56B is set larger than the distance D1 betweenthe first condenser lens 55A and the first aperture plate 56 so thatonly the fluorescence K1 can pass through the aperture of the firstaperture plate 56A and only the fluorescence K2 can pass through theaperture of the second aperture plate 56B, whereby the fluorescence K1and the fluorescence K2 are separately detected by the photomultipliers40A and 40B, respectively.

Operation of the image information read-out system of this embodimentwill be described, hereinbelow.

When the laser beams L1 and L2 are projected onto the upper and lowersurfaces of the test piece 1, fluorescence K1 and fluorescence K2 areemitted from the upper and lower surfaces of the test piece 1,respectively, and simultaneously travel in the direction of arrow X as alight bundle. The light bundle is divided into two light bundles by thehalf-silvered mirror 63, one traveling toward the first photomultiplier40A and the other traveling toward the second photomultiplier 40B. Thefluorescence K1 included in said one light bundle impinges upon thefirst photomultiplier 40A through the first condenser lens 55A, thefirst aperture plate 56A and the first laser beam cut filter 41A and isdetected by the first photomultiplier 40A, whereas the fluorescence K2included in said the other light bundle impinges upon the secondphotomultiplier 40B through the second condenser lens 55B, the secondaperture plate 56B and the second laser beam cut filter 41B and isdetected by the second photomultiplier 40B.

The fluorescence K1 and the fluorescence K2 are photoelectricallyconverted to electric signals respectively by the first and secondphotomultipliers 40A and 40B, and the electric signals are amplified bythe first and second amplifiers 42A and 42B and then digitized by thefirst and second A/D converters 43A and 43B. Then visible images aredisplayed on a monitor (not shown) on the basis of the digitizedelectric signals.

Thus also in the image information read-out system of this embodiment,image information can be read out from opposite sides of the test piece1 of the first embodiment of the present invention, where cDNAs aredisposed on opposite sides of the substrate 2.

The image information read-out system of this embodiment can be used toread out image information from the test piece of the second embodimentof the present invention shown in FIG. 3. In this case, positions of thefirst and second lenses 31A and 31B and the first and second apertureplates 56A and 56B are adjusted to read out image information from thesubstrate segments 2A and 2B.

Though, in the embodiments described above, the CDNAs are disposed notto overlap each other in the direction of thickness of the substrate 2or the substrate segments 2A and 2B, they may overlap each other in thedirection of thickness of the substrate 2 or the substrate segments 2Aand 2B when the image information read-out system comprises a confocaloptical system.

What is claimed is:
 1. A test piece for use in biological analysescomprising: a planar substrate having an upper surface and a lowersurface; a plurality of different known specific binding substancesdisposed in predetermined positions on said substrate; wherein thespecific binding substances are disposed on said upper surface and onsaid lower surface of said substrate so as not to be within saidsubstrate, the specific binding substances being disposed such that theydo not contact one another on each said upper surface and said lowersurface, and wherein the specific binding substances are disposed onsaid upper surface and on said lower surface of said substrate such thatthe specific binding substances on said upper surface do not have thespecific binding substances on said lower surface disposed directlybeneath them when said test piece is viewed in a direction of thicknessof said substrate.
 2. A test piece for use in biological analysescomprising: a plurality of planar substrates each having an uppersurface and a lower surface, said substrates being stacked and bondedtogether in a vertical direction with a spacer disposed between thesubstrates; a plurality of different known specific binding substancesdisposed in predetermined positions on said upper surface of each ofsaid plurality of substrates so as not to be within any individual oneof said plurality of substrates; wherein the specific binding substancesare disposed such that they do not contact one another on said uppersurface of each of said plurality of substrates; and wherein thespecific binding substances are disposed such that the specific bindingsubstances on one of said plurality of substrates are not disposeddirectly beneath the specific binding substances on another of saidplurality of substrates when said test piece is viewed in a direction ofthickness of said substrate.
 3. The test piece of claim 1, wherein saidplanar substrate is positioned between the specific binding substances.4. A test piece for use in biological analyses comprising: a pluralityof planar substrates each having an upper surface and a lower surface,said substrates being stacked and bonded together in a verticaldirection with a spacer disposed between the substrates, such that thestacked substrates are parallel to one another; a plurality of differentknown specific binding substances disposed in predetermined positions onsaid upper surface of each of said plurality of substrates so as not tobe within any individual one of said plurality of substrates; whereinthe specific binding substances are disposed such that they do notcontact one another on said upper surface of each of said plurality ofsubstrates; and wherein the specific binding substances are disposedsuch that the specific binding substances on one of said plurality ofsubstrates are not disposed directly beneath the specific bindingsubstances on another of said plurality of substrates when said testpiece is viewed in a direction of thickness of said substrate.
 5. Thetest piece of claim 1, wherein said planar substrate is opticallytransparent.
 6. The test piece of claim 2, wherein said planar substrateis optically transparent.